It is well known in the art that DNA as well as RNA can be methylated. The base 5-methylcytosine is the most frequent covalently modified base found in the DNA of eukaryotic cells. DNA methylation plays an important biological role in, for example, regulating transcription, genetic imprinting, and tumorigenesis (for review see, e.g., Millar et al.: Five not four: History and significance of the fifth base; in The Epigenome, S. Beck and A. Olek (eds.), Wiley-VCH Publishers, Weinheim 2003, pp. 3-20). The identification of 5-methylcytosine is of particular interest in the area of cancer diagnosis. But the identification of methylation is difficult. Cytosine and 5-methylcytosine have the same base-pairing behavior, making 5-methylcytosine difficult to detect using particular standard methods. The conventional DNA analysis methods based on hybridization, for example, are not applicable. In addition, the methylation information is lost completely by the amplification by means of PCR.
Accordingly, current methods for DNA methylation analysis are based on two different approaches. The first approach utilizes methylation specific restriction enzymes to distinguish methylated DNA, based on methylation specific DNA cleavage. The second approach comprises selective chemical conversion (e.g., bisulfite treatment; see e.g. WO 2005/038051) of unmethylated cytosines to uracil while methylated cytosines remain unchanged. Uracil has the same base pairing behavior as thymine. It therefore forms base pairs with adenine. Instead, 5-methylcytosine hybridizes with guanine still after bisulfite treatment. It is therewith possible to differentiate between methylated and unmethylated cytosines. The enzymatically or chemically pretreated DNA generated in these approaches is typically pre-amplified and analyzed in different ways (see, e.g., WO 02/072880 pp. 1 ff; Fraga and Estella: DNA methylation: a profile of methods and applications; Biotechniques, 33:632, 634, 636-49, 2002). The pre-amplification of chemically pretreated DNA leads to an enhanced sensitivity of the subsequent detection reaction.
Different PCR methods are known in the art for analyzing converted and unconverted cytosine positions. Selective amplification only of unconverted (methylated) or with the reverse approach, converted (unmethylated) cytosine positions is attained by using methylation specific primers in so-called methylation-specific PCR (MSP) methods, or by using ‘blockers’ in “HeavyMethyl™” methods (see, e.g., Herman et al.: Methylation specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 93:9821-6, 1996; Cottrell et al.: A real-time PCR assay for DNA-methylation using methylation specific blockers. Nucl. Acids Res., 32:e10, 2004). Alternatively, it is possible to amplify the DNA in a non-methylation specific manner, and analyze the amplificates by means of methylation specific probes (see, e.g., Trinh et al.: DNA methylation analysis by MethyLight technology. Methods, 25:456-62, 2001). Particular PCR-based methods are also applicable as ‘real-time’ PCR variants, making it possible to detect the methylation status directly in the course of the PCR, without the need for a subsequent analysis of the products (MethyLight™; WO 00/70090; U.S. Pat. No. 6,331,393; and Trinh et al. 2001, supra).
Quantification of the degree of DNA methylation is required in many applications including, but not limited to, classification of tumors, obtaining prognostic information, or for predicting drug effects/responses. Different methods of such quantification are known in the art, such as ‘end-point analysis’ and ‘threshold-value analysis’.
End-point, analyses: To some extend, the DNA is pre-amplified, like for example in the Ms-SNuPE method, for the hybridization on microarrays, for hybridization assays in solution or for direct bisulfite sequencing (see, e.g., Fraga and Estella 2002, supra). A problem with such “end point analyses” (where the amplificate quantity is determined at the end of the amplification) is that the amplification can occur non-uniformly because of, inter alia, obstruction of product, enzyme instability and/or a decrease in concentration of the reaction components. Correlation between the quantity of amplificate, and the quantity of DNA utilized is, therefore, not always suitable, and quantification is thus sensitive to error (see, e.g., Kains: The PCR plateau phase—towards an understanding of its limitations. Biochem. Biophys. Acta 1494:23-27, 2000).
Threshold-value analyses: By contrast, threshold-value analysis, which is based on a real-time PCR, determines the quantity of amplificate in the exponential phase of the amplification, rather than at the end of the amplification. Such threshold, real-time methods presume that the amplification efficiency is constant in the exponential phase. The art-recognized threshold value ‘Ct’ is a measure corresponding, within a PCR reaction, to the first PCR cycle in which the signal in the exponential phase of the amplification is greater than the background signal. Absolute quantification is then determined by means of a comparison of the Ct value of the investigated (test) DNA with the Ct value of a standard (see, e.g., Trinh et al. 2001, supra; Lehmann et al.: Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol., 160:605-12, 2002). A substantial problem of such Ct value-based analyses is that when high DNA concentrations are used, only a small resolution can be achieved. This problem also applies when high degrees of methylation are determined via PMR values (for discussion of PMR values see, e.g., Eads et al., CANCER RESEARCH 61:3410-3418, 2001.) Additionally, amplification of a reference gene (e.g., the β-actin gene) is also required for this type of Ct analysis (see, e.g., Trinh et al. 2001, supra). (An overview of real time PCR based quantification can be obtained from WO 2005/098035; Real-Time PCR: An Essential Guide, Horizon Bioscience, Kirstin Edwards, Julie Logan and Nick Saunders, May 2004 ISBN: 0-9545232-7X; Real-time PCR, M. Tevfik Dorak, Taylor & Francis, April 2006), ISBN: 041537734X; Mackay I M, Arden K E, Nitsche A. Real-time PCR in virology. Nucleic Acids Res. 2002 Mar. 15; 30(6):1292-305; Bernard P S, Wittwer C T. Real-time PCR technology for cancer diagnostics. Clin Chem. 2002 August; 48(8):1178-85; Bernhard Kaltenboeck and Chengming Wang. Advances in real-time PCR: Application to clinical laboratory diagnostics. Advances in Clinical Cancer, 2005; 40:219-259).
A critical parameter for methylation analysis is sensitivity. The reason for this is the problem that samples to be analyzed usually comprise heterogeneous DNA. The DNA is of the same sequence but has a different methylation. Thereby, DNA with a sought methylation pattern is only present in low amounts. An example is the tumor diagnosis out of body fluids. The death of tumor cells results in a release of tumor DNA into body fluids like blood. But also the DNA of died healthy cells is found in the blood. Various levels of tumor DNA are found besides non-tumor DNA depending on the size and the progression of the cancer disease. Because of obvious reasons, an early as possible detection of a tumor is favorable. This means that the slightest amount of tumor DNA has to be reliable detected and correctly analyzed during methylation analysis. As more sensitive a method for methylation analysis is as more early tumor DNA can be detected and a tumor can be diagnosed.
Another example is the detection of a cell type by detection of its specific methylation in a biopsy sample comprising various cell types. Thereby, the presence or absence of said cell type may be indicative for a disease or for a likely respond to a treatment. Also in this case the slightest amount of cell type specific DNA has to be reliable detected and correctly analyzed by methylation analysis
Therefore it is a major concern in the field of the art exists to improve the sensitivity of known methods for methylation analysis or to provide new methods with a high as possible sensitivity.
The method for methylation analysis with the so far highest specificity is the real time QM method (quantitative methylation method; WO 2005/098035). Here a non-methylation specific, conversion specific amplification of the target DNA is performed. The amplificates are detected by means of the hybridization of two different methylation specific real-time PCR probes. Thereby one of the probes is specific for the methylated state, while the other probe is specific for the unmethylated state. The two probes bear different fluorescent dyes. A quantification of the degree of methylation is obtained within specific PCR cycles employing the ratio of signal intensities of the two probes. Alternatively, the Ct values of two fluorescent channels can also be drawn on for the quantification of the methylation.
Because of obvious reasons, another major concern in the art exists in providing methylation analysis methods which ensure a high as possible sensitivity. This means for example that as much as possible of all samples derived from individuals having cancer are detected within a group of samples derived from individuals having cancer or not.
The method for methylation analysis with the so far highest specificity is an embodiment of the above mentioned HeavyMethyl™ method in which methylation specific blockers and probes are used in real time PCR. Thereby the blocker is specific for certain unmethylated cytosine position(s) while the probe is specific for the same cytosine position(s) being methylated, or vice versa.
Currently the applicant is not aware of any method with a greater specificity as the QM method or a greater sensitivity as the HM method.